Generally, stable water soluble purified polypeptides which are potent inhibitors of endothelial cell proliferation in-vivo may be useful in elucidating the mechanism by which angiogenesis is regulated. Specifically, stable water soluble polypeptide inhibitors of angiogenesis reduce the volume of animal tumors, including human tumors, in-vivo.
Evidence suggests that angiogenesis is essential for the growth and persistence of solid tumors. Folkman, 1989; Hori et al., 1991; Kim et al. 1993. To stimulate angiogenesis, tumors up regulate their production of a variety of angiogenic factors, including the fibroblast growth factors. Kandel et al., 1991. Studies suggest that proteins such as murine endostatin protein (184 residue protein derived from the cleavage of Type XVIII collagen expressed by hemanioendothelioma EOMA or obtained as an expression product from recombinant cells) may cause marked reduction of mouse tumors. U.S. Pat. No. 5,854,205, hereby incorporated by reference. Similarly, angiostatin protein (200 residue protein derived from the cleavage of plasminogen) has been shown to inhibit the metastasis of certain primary mouse tumors. http://www.ultranet.com/jkimball/BiologicalPages/A/Angiogenesis.html (Mar. 31, 2000), hereby incorporated by reference.
The potential use of the entire endostatin and angiostatin proteins as human drug therapies has been surrounded by enormous publicity and there appears to be a large commercial market for endostatin or angiostatin proteins, or other angiogenesis inhibitors as drugs. Because of the growing commercial, therapeutic, and research potential for angiogenesis inhibitors, numerous research studies have been conducted which disclose a variety of uses for the endostatin and angiostatin proteins. In spite of the numerous studies conducted with whole endostatin and angiostatin proteins, substantial problems remain unresolved with regard to providing angiogenesis inhibitors that can be commercialized, or used as drug therapies in animals or in humans, or as angiogenic compounds in research.
A significant problem with whole protein angiogenesis inhibitors, for example endostatin proteins or angiostatin proteins, can be that it may be difficult to predict what portion of the protein is biologically active. As disclosed by U.S. Pat. No. 5,854,205, hereby incorporated by reference, the endostatin protein is comprised of 184 residues with a molecular weight of about 20 kDa. Similarly, the angiostatin protein comprised of 200 residues has a molecular weight of about 21 kDa. With respect to such protein angiogenesis inhibitors, it is difficult to predict which residues encompassed by the primary structure of the protein may be responsible for the observed angiogenesis inhibition activity. One aspect of this difficulty may be that the biologically active portion of the protein may comprise discontinuous regions of the primary structure of the protein which must be held in a specific secondary or tertiary structure by the remaining portions of the protein molecule to acquire angiogenesis inhibition activity. A second aspect of this difficulty may be that the biologically active region of the protein molecule may be a continuous region of the primary structure of the protein which must be similarly held in a specific secondary or tertiary structure by the remaining portion of the protein molecule to acquire angiogenesis inhibition activity. A third aspect of this difficulty may be that there may be a plurality of biologically active regions encompassed by the primary sequence of the protein some of which may be discontinuous or some of which may be continuous. A fourth aspect of this difficulty may be that such plurality of biologically active regions encompassed by the primary sequence of the protein overlap one another and may not be independently excised from the primary sequence with out disabling the other biologically active regions. A fifth aspect of this difficulty may be that the region of a protein having angiogenesis inhibition activity may be discontinuous from the region of the protein which has an affinity for the target cell receptor. A sixth aspect of this difficulty may be that a portion of an angiogenic protein may apparently lack biological activity when assayed in-vitro but may acquire biological activity when assayed in-vivo. A seventh aspect of this difficulty may be that a portion of an angiogenic protein when chemically or enzymatically excised, or when identified and subsequently chemically synthesized, may not be biologically available to the target receptor in-vitro or in-vivo. This lack of biological availability may be due to insolubility of the compound, a binding affinity to surrounding substrates that is greater than to the target cell receptor, instability of the angiogenic compound with respect to cleavage, or with respect to modification of the peptide backbone, N-terminus, C-terminus, side chain, or other peptide or chemical moiety associated with the excised or chemically synthesized portion of the protein. Due to these, and a variety of other difficulties well known to those with skill in the art, assignment of angiogenesis inhibition activity to any specific biochemical structure, which may be a portion of a protein, such as endostatin or angiostatin, or any other molecule, may be unpredictable without an actual reduction to practice involving at least isolation, purification, and in-vitro and in-vivo assays to confirm biological activity of a particular compound. Subsequent characterization and identification of the chemical structure may further serve to differentiate biologically active compounds which in every other respect may seem similar.
Another significant problem with the commercial development of additional novel angiogenesis inhibitors may be that small polypeptides (primary sequences comprised of 65 or fewer amino acid residues) have not been shown to have angiogenesis inhibition activity. One aspect of this problem may be due to the failure to incorporate within the primary sequence of the polypeptide the essential residues which have an affinity for the target cell receptor. A second aspect of this problem may be the failure to incorporate within the primary sequence of the polypeptide the essential residues which comprise the region conferring angiogenesis inhibition activity to the polypeptide. A third aspect of this problem may be that in-vitro methods of assaying polypeptides for biological activity may fail to properly address the processing requirements of the polypeptide in a manner which in-vivo methods for assaying polypeptides do address.
Another significant difficulty with existing angiogenesis inhibitors may be that therapeutic results are difficult to replicate. With respect to endostatin protein, for example, it can be difficult to reproduce results which show endostatin protein dramatically shrinks tumors. Harvard Cancer Research Questioned, New York Times Company, Associated Press (1998); Ovarian Cancer Research Notebook http://www.slip.net/mcdavis/database/angio188.htm; National Cancer Institute Clarifies Role in Development of Endostatin, Angiogenesis Weekly, (Tuesday, Oct. 5, 1999), each hereby incorporated by reference.
Another significant problem with existing angiogenesis inhibitors may be stability. Some angiogenesis inhibitors have proven to be unstable in shipment, or during subsequent routine handling, or during routine use in research studies resulting in a partial or complete loss of biological activity. National Cancer Institute Clarifies Role in Development of Endostatin, Angiogenesis Weekly, (Tuesday, Oct. 5, 1999), hereby incorporated by reference.
Another significant problem with existing angiogenesis inhibitors may be that it is difficult to produce sufficient amounts for wide spread use either for research or for therapies. Recombinant unfolded liner endostatin protein, for example, is insoluble (as disclosed by U.S. Pat. No. 5,854,205, hereby incorporated by reference) and may be difficult to use in applications which require soluble protein or soluble portions thereof, such as isotopic labeling, affinity purification substrates for receptor proteins, competing other polypeptides or proteins in solution, for use in diagnostic kits for detecting the presence of antibodies, or for use as conventional drug therapies in animals or humans. The difficulty in producing large quantities of some types of angiogenesis inhibitors, including protein angiogenesis inhibitors, may present significant obstacles in developing vascularization inhibition therapy models.
Another significant problem with existing angiogenesis inhibitors may be that the effective dosages are too high. For example, the amount of angiostatin protein used in animal studies has been criticized as being too high for clinical trials in humans. Annual Review of Medicine, 49: 407-424, (1998); and http://www.lef.org/protocols/abstracts/cancertreat-abstr.html, each hereby incorporated by reference. One aspect of high dosages with respect to conventional protein angiogenic inhibitors may be that a substantial portion of the protein by weight does not contribute to the observed angiogenic inhibition activity. A second aspect of high dosages with respect to conventional protein angiogenic inhibitors may be that the angiogenic inhibition activity on a molar basis may be lower than is practical for a particular application. A third aspect of high dosages with respect to conventional protein angiogenic inhibitors may be that the protein angiogenic inhibitors are unstable with respect to proteolytic activity, temperature, handling, or methods of in-vitro or in-vivo assays, or may have other attributes such as insolubility, a processing requirement, or high elimination rates in-vivo, as examples, which may render the active portions of conventional angiogenic inhibitors biologically unavailable or at levels which are not practical for applications such as human drug therapy.
Another significant problem with existing angiogenesis inhibitors may be that they are not suitable for use in humans. One aspect of this problem can be that such angiogenesis inhibitors comprise proteins derived from species other than human. For example murine endostatin (derived from mouse tissue, mouse cell lines, or recombinant expression of mouse endostatin genes) which has shown to be effective in regulating endothelial cell growth in mice or effective in reducing the volume of mouse tumors may not be applicable to the regulation of endothelial cell growth in humans or for the reduction in volume of human tumors grown in mouse or for the reduction of tumor volume in humans. See for example, U.S. Pat. No. 5,854,205, hereby incorporated by reference.
Another significant problem with existing angiogenesis inhibitors may be solubility. One aspect of this problem may be that angiogenic inhibitor proteins become denatured during isolation or purification and may subsequently become insoluble as such proteins unfold presenting additional hydrophobic core residues. As disclosed by U.S. Pat. No. 5,854,205, hereby incorporated by reference, unfolded recombinant mouse endostatin is not soluble and may not be tested in-vitro. A second aspect of this problem may be that only a small percentage of the unfolded protein may spontaneously refold, and an even smaller percentage of the protein molecules may refold properly. Unfolded or improperly folded molecules may have no or lower biological activity on a weight or molar basis than the properly folded proteins. A third aspect of this problem may be that unfolded insoluble angiogenic proteins may have to be injected as a suspension of particulate. This may make the preparation of the appropriate dosage more difficult or more time consuming. Moreover, reabsorption of injected precipitates may be variable from individual to individual or injection site to injection site. It is well known to those with skill in the art that injecting insoluble suspensions of proteins, peptides, or protein-peptide conjugates into animals often stimulates the production of antibodies to such insoluble particulate, however, the results of immunization of animals with such insoluble particulate may not be predictable even when using what appears to be the same amount of suspended particulate.
Another problem with generating additional novel angiogenesis inhibitors may be that compounds which may fail to show inhibition of angiogenic activity in-vitro may show unexpectedly high levels of angiogenic inhibition activity in-vivo. This discovery teaches away from conventional wisdom of those skilled in the art. Up until the present invention it was thought that xe2x80x9cproteins or peptides derived from . . . sources including manual or automated synthesis, may be quickly and easily tested for endothelial proliferation inhibiting activity using a biological assay such as the bovine capillary endothelial cell proliferation assay.xe2x80x9d See U.S. Pat. No. 5,854,205, hereby incorporated by reference. However, it is now understood that stable water soluble polypeptides may show no effect in-vitro but still retain the ability to reduce the volume of established xenographs in-vivo. J. D. Hunt et al., Internal Peptides Within Endostatin Lacking Zinc-Binding Domains Inhibit Angiogenesis, Publishing ID: 3106, Meeting of the American Cancer Society (Apr. 4, 2000), hereby incorporated by reference. As generally mentioned above, and specifically with respect to endostatin protein, it is unpredictable and difficult to assess which portions of a protein may encompass observed biologically active. Moreover, minor chemical modifications, such as the reduction in the number of residues, or the amidation of the C-terminus of a polypeptide, for example, may confer unexpected properties upon a polypeptide such as increased biological activity, increased stability, or increased solubility. As such, no change in the structure of a protein or of a polypeptide or other compound, regardless as to how minor it may be perceived before hand, should be considered silent until proven so by the appropriate testing in-vitro and in-vivo as disclosed below.
Another problem with generating additional novel angiogenesis inhibitors may be that the molecular structure may not be chemically synthesized in substantial quantity. Angiogenesis inhibiting proteins such as endostatin (184 residues) and angiostatin (200) may not be able to be chemically synthesized with any substantial success. Conventional methods of chemical synthesis resulting in substantial quantities of purified peptide (over 10 milligrams) are typically limited to peptide sequences having fewer than 70 residues. Peptides which have fewer than 35 residues may be candidates for the chemical synthesis of large amounts of synthetic purified peptide in the kilogram quantities. Peptides and proteins have a greater number of residues, are generally produced by recombinant technologies. Although shorter polypeptides may also be produced by recombinant technology, chemical synthesis of shorter polypeptides may be less expensive, less time consuming, and result in a more highly purified end product. Moreover, conventional methods of chemical synthesis of polypeptides are available (either in-house or commercially) to a broader range of companies and research individuals than recombinant techniques.
Another problem with generating additional novel angiogenesis inhibitors may be that both the human and mouse counterparts of the same angiogenic inhibitor may not be available. Having both the mouse and human counter parts of the same angiogenic inhibitor allows for a animal model in which angiogenic inhibitors may be researched and the findings more readily extrapolated to similar experimental trials with humans.
With respect to making and using cell growth regulators, and specifically with respect to angiogenic inhibitors the present invention discloses compositions and technology which address every one of the above-mentioned problems in a practical fashion.
A broad object of embodiments of the invention can be to provide potent polypeptide inhibitors of angiogenesis and methods for their use. Polypeptide inhibitors of angiogenesis may be important in elucidating the process of vascularization which involves both positive and negative regulators. Vascularization which is induced by pro-angiogenic factors may be balanced using various embodiments of the invention which may include negative regulators of angiogenesis.
Another broad object of embodiments of the invention can be to provide polypeptide inhibitors of angiogenesis that can be labeled isotopically, or with other molecules, or with other proteins for use in the detection and visualization of binding sites for negative regulation of angiogenesis with state of the art techniques such as positron emission tomography, autoradiography, flow cytometry, radioreceptor binding assays, competition assays, immunochemistry, or the like.
Another significant object of an embodiment of the invention can be to provide polypeptide inhibitors of angiogenesis which may be used to identify, isolate, or purify receptor proteins which bind to such polypeptide inhibitors along with the methods for reversibly associating the receptor proteins with the polypeptide invention. These receptor proteins may include the human receptor protein which binds the human endostatin protein.
Another significant object of an embodiment the invention can be to provide polypeptide inhibitors of angiogenesis which may be used to produce monoclonal or polyclonal antibodies which have an affinity to such polypeptide inhibitors or to the endostatin protein or portions thereof.
Another object of an embodiment of the invention can be to provide polypeptide inhibitors of angiogenesis which may be used as agonists or antagonists of the endostatin protein or a portion of the endostatin protein.
Another object of an embodiment of the invention can be to provide polypeptide inhibitors of angiogenesis for use in diagnostic or prognostic methods or kits for detecting the presence and quantity of antibodies which bind endostatin protein or a portion thereof in biological fluids. Kits and the methods for detecting such antibodies can be in various configurations well known to those of ordinary skill in the art. One aspect of this embodiment of the invention may be to provide a method for the detection of or prognosis of cancer.
Another significant object of an embodiment of the invention can be to provide polypeptide inhibitors of angiogenesis as a therapy for treating diseases and processes that are mediated by angiogenesis. Such diseases and processes, may for example include, but are not limited to, preventing tumor growth, inhibiting tumor growth, reduction in tumor volume, elimination of tumors, inhibiting metastasis, preventing metastasis, inducing dormancy of tumors and metastasis, preventing arteriosclerosis, inhibiting atherosclerosis, reducing existing plaque in blood vessels, reducing intimal plaque neovascularization, hemangioma, solid tumors, leukemia, myocardial angiogenesis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularizaton, macular degeneration, wound healing, peptic ulcer, fractures, keloids, hematopoiesis, ovulation, menstruation, or placentation.
Another object of an embodiment of the invention can be to provide polypeptide inhibitors of angiogenesis for in-vivo use without acquired drug resistance.
Another object of an embodiment of the invention can be to provide polypeptide inhibitors of angiogenesis for in-vivo use which produce minimal side effects.
Another object of an embodiment of the invention can be to provide polypeptide inhibitors which require in-vivo processing to acquire cell growth regulation activity.
Yet another object of an embodiment of the invention may be to provide polypeptides which may be used to detect and quantify endostatin biosynthesis.
Still another significant object of an embodiment of the invention can be can be to provide polypeptide inhibitors of angiogenesis for delivering cytotoxic molecules to receptor targets for therapeutic or research purposes.
Another object of an embodiment of the invention is to provide a composition comprising antibodies. One aspect of this objective may be to provide either monoclonal, or polyclonal, or mixtures thereof, to polypeptide inhibitors of angiogenesis, or to corresponding regions of the endostatin protein. A second aspect of this objective may be to provide either monoclonal, or polyclonal, or mixtures thereof which inhibit binding of such monoclonal, or polyclonal antibodies to polypeptide inhibitors of angiogenesis.
Another significant object of an embodiment of the invention can be can be to provide polypeptide inhibitors of angiogenesis which can be synthesized chemically which may obviate the use of enzymatic cleavage of endostatinprotein or the products of recombinant technologies for production of such polypeptide angiogenesis inhibitors.
Another object of an embodiment of the invention can be to provide polypeptides which bind to receptor proteins which are associated with regulation of angiogenesis, but other than competing with other polypeptides or proteins for the same receptor binding site, do not directly regulate cell growth. One aspect of this object may be to provide polypeptides which comprise only a binding region to compete other molecules which bind to the same receptor site. A second aspect of this object may be to provide polypeptides that have a binding region and a cell growth regulation region. With regard to this second aspect, the cell growth regulation region may be inactive in-vitro but active in-vivo, or may be active in-vitro but inactive in-vivo, or may be active in-vitro and active in-vivo.
Another object of an embodiment of the invention can be to provide polypeptide angiogenesis inhibitors which regulate the growth endothelial cells in-vivo at low dosages. One aspect of this object of the invention may be to use smaller amounts of such polypeptide by weight or by moles than the amount of endostatin protein used by weight or by moles with similar or enhanced therapeutic results in treatment of the same disease.
Another object of an embodiment of the invention can be to provide polypeptides which inhibit the growth of human prostrate cancer tumors or cells in-vivo.
Another object of an embodiment of the invention can be to provide polypeptide inhibitors of angiogenesis which may be stable in aqueous solutions or water.
Another object of an embodiment of the invention can be to provide polypeptide inhibitors of angiogenesis which correspond to a portion of the human endostatin protein. One aspect of this object is to provide such polypeptides which are not the N-terminal portion of the endostatin protein. A second aspect of this object may be to provide such human polypeptide from the internal region of the human endostatin protein.
Another object of an embodiment of the invention can be to provide polypeptide inhibitors of angiogenesis which correspond to a portion of the mouse endostatin protein. An aspect of this object is to provide such mouse polypeptide from the internal region of mouse endostatin protein. A second aspect of this object is to provide such mouse polypeptides which correspond to similar regions of the human endostatin molecule.
Another object of an embodiment of the invention can be to provide methods of enzymatic cleavage of human and mouse endostatin which generate polypeptides which inhibit angiogenesis.
Another object of an embodiment of the invention can be to provide a stable, water soluble, 63-residue polypeptide inhibitor of angiogenesis.
Another object of an embodiment of the invention can be to provide a stable, water soluble, 31-residue poly peptide inhibitor of angiogenesis.
Another object of an embodiment of the invention can be to provide methods of chemical synthesis of a nested set of polypeptides or analogs from an internal regions of the human or mouse endostatin protein.
These and other independent objects, features, and advantages of the invention are disclosed throughout other areas of the specification.